The work accomplished during this year falls into two categories. The first represents a continuation of our investigation into the enzymatic mechanisms responsible for the production of met5-enkephalin in adrenal chromaffin cells. We have further characterized a trypsin-like serine protease present in adrenal chromaffin granules by studying reactivity of this enzyme toward proenkephalin-derived peptides purified from chromaffin granules. Cleavage at pairs of basic vs. single basic amino acids was assessed using the 5.3 K dalton fragment as a substrate. The enzyme cleaved this peptide after a pair of basic amino acids, thus generating met5-enk-arg6-gly7-leu8 (MERGL); no cleavage at the single arginine in MERGL was observed. Thus the enzyme exhibits limited specificity which would be expected for a prohormone processing enzyme. Purified human HMW and LMW kininogen were also used as substrates for the enzyme; no cleavage of kininogens to bradykinin was observed, suggesting that human kininogens are not recognized by this enzyme. In addition the release of the various molecular weight forms of the octapeptide MERGL from brain tissue was studied. Brain slices prepared from areas which were known from previous work to contain both high and low molecular weight forms of MERGL-IR peptides were found to release both of these forms following stimulation with high potassium. Gel filtration of released immunoreactivity revealed that 63% of the IR released from slices of rat medulla-pons was in the high molecular weight form. Approximately half of the immunoreactive molecules released from slices of rat hypothalamus were present as the high molecular weight MERGL-IR peptide. These results suggest further investigation directed to ascertain whether this peptide functions as a neuromodulator in addition to its role as a precursor for MERGL.